The NCp7 zinc fingers of the HIV-1 nucleocapsid protein are an excellent target for the development of new antiviral agents due to their structural conservation and broad range of function that includes integration, reverse transcription, Gag processing, and viral maturation. Previously, we found that a number of compounds of the S-acyl 2-mercaptobenzamide thioester (SAMT) chemotype altered NCp7 functions and were shown to be broadly anti-retroviral. During the past year we gained important insights into the mechanism of action of these compounds. We treated HIV-1-infected H9 cells with a series of SAMTs to examine how these compounds affect Gag particle production and processing. They did not appear to appreciably decrease particle formation, but virions from treated cells contained mostly unprocessed Gag and were non infectious. Based on structure-activity relationships observed with this series of compounds, we determined that the ortho position for the sulfur on the benzamide ring in the SAMT structure is critical for these effects, likely due to the formation of a stable intramolecular hydrogen bond. Consistent with this idea, positional changes or substitutions made at these sites disrupt the compound's antiviral activity. Recent studies have demonstrated that NCp7 Cys39 of the C-terminal zinc finger is covalently modified by an active SAMT, resulting from the transfer of the acyl moiety the thioester to this cysteine side chain and the production of a free thiol leaving group that may be reacylated within the cell by the action of acyl-CoA. To demonstrate this reaction in a model system, we treated chronically infected H9 cells with SAMT-247 that contains a [14]C-radiolabeled acyl group. The virus particles isolated from treated cells contained radiolabeled p55Gag as observed by SDS-PAGE. NMR structure studies using recombinant NCp7 confirmed these results. Recently, we have analyzed the interactions of two mercaptobenzamide thioesters with structural binding domains (ZBDs) from six different proteins. These six proteins represent three different classes (Cys2His2, Cys3His, and Cys4) of ZBDs, with five distinct structural motifs. The results demonstrate that the thioester compounds show reactivity towards specific classes of structural ZBDs found in cellular proteins. Together, our studies suggest that SAMTs covalently modify NCp7 through a transacylation reaction resulting in zinc ejection and loss of structure and function. In our ongoing medicinal chemistry efforts, we studied the potential of SAMTs as components in topical microbicides. We designed and tested three SAMT thioesters that were shown to be virucidal and to inhibit cell-to-cell associated transmission of HIV in coculture systems. The compounds inhibited the DC-SIGN mediated transmission of virus without interfering with the binding of HIV to DC-SIGN in Raji cells transfected to express DC-SIGN. The compounds were also assessed ex vivo in human cervicovaginal tissue explants and prevented dissemination of HIV via mucosal migratory cells (dendritic cells, lymphocytes and macrophages), without altering explant or epithelial viability or proinflammatory cytokine secretion. Finally, we determined the in vivo effects of SAMT thioesters on infectivity of HIV particles in a murine HIV transgenic model and of SIV in Chinese rhesus macaques. Infectivity of both HIV and SIV were significantly reduced following continuous alzet pump delivery of 0.4 mg/kg and 2.2 mg/kg SAMT to mice and monkeys, respectively in the absence of systemic or local toxicity.